Beta-lactamase inhibitors, formulations, and uses thereof

ABSTRACT

Provided herein are heterocyclic compounds and pharmaceutical formulations that can be used to treat bacterial infections. Also provided herein are methods of making and using the heterocyclic compounds and pharmaceutical formulations.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of co-pending PCT Patent ApplicationNo. PCT/US2017/021905, filed on Mar. 10, 2017, entitled “BETA-LACTAMASEINHIBITORS, FORMULATIONS, AND USES THEREOF,” the contents of which isincorporated by reference herein in its entirety.

PCT Patent Application No. PCT/US2017/021905 claims the benefit of andpriority to U.S. Provisional Patent Application No. 62/307,013, filed onMar. 11, 2016, entitled “BETA-LACTAMASE INHIBITORS, FORMULATIONS, ANDUSES THEREOF,” the contents of which is incorporated by reference hereinin its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant number R01AI103158 awarded by the National Institutes of Health. The Governmenthas certain rights in the invention.

BACKGROUND

Beta-lactam antibiotics are among the most commonly prescribedantibiotics. Beta-lactam antibiotics target the cross-linking of thebacterial cell wall, which can ultimately result in cell death. Despitethe numerous successes of the beta-lactam antibiotics, bacteria havedeveloped resistance to them. As such there exists a need for improvedantibacterial treatments to overcome bacterial resistance to beta-lactamantibiotics.

SUMMARY

In some aspects, provided herein are compositions according to Formula 4

wherein R₂ can be selected from the group of H, CH₃, Cl, Br, F, OCH₃,CF₃, an alkyl, a cycloalkyl, and an aryl, and wherein R₄ can be selectedfrom the group of H, CH₃, Cl, Br, F, OCH₃, CF₃, an alkyl, a cycloalkyl,and an aryl. In some aspects, R₂ can be H and R₄ can be H.

Also provide herein are pharmaceutical formulations containing an amountof a composition according to Formula 4, wherein R₂ can be selected fromthe group of H, CH₃, Cl, Br, F, OCH₃, CF₃, an alkyl, a cycloalkyl, andan aryl, and wherein R₄ can be selected from the group of H, CH₃, Cl,Br, F, OCH₃, CF₃, an alkyl, a cycloalkyl, and an aryl or wherein R₂ canbe H and R₄ can be H. The pharmaceutical formulations can furtherinclude a beta lactam antibiotic. The amount of a composition accordingto Formula 4 can be an effective amount. The effective amount canincrease the efficacy of a beta lactam antibiotic and/or reduce theamount of or the activity of a beta lactamase.

Also provided herein are methods of treating a subject in need thereof,wherein the method can include the step of administering an amount of acomposition according to Formula 1

wherein X₁ can be O, O⁻, CH, or N—R₈, wherein R₈ can be H, an alkyl; ora heteroalkyl; wherein X₂ can be PO(OH)₂, SO(OH)₂, CONHOH, COOH, orN(OH)COR₆, where R₆ can be H or CH₃; wherein X₃ can be H or SH; whereinX₄ can be CH, C, NH, or N⁺; wherein X₅ can be CH, or NH; wherein R₁ canbe H, CH₃, Cl, Br, F, OCH₃, CF₃, an alkyl, a cycloalkyl, or an aryl;wherein R₂ can be H, CH₃, Cl, Br, F, OCH₃, CF₃, an alkyl, a cycloalkyl,aryl, or R₂ and R₃, when taken together with the atoms to which they areattached, can form a cyclic structure having 5 or 6 carbon atoms, one ormore of which can be a heteroatom; wherein R₃ cam be H, CH₃, Cl, Br, F,OCH₃, CF₃, an alkyl, a cycloalkyl, or an aryl; wherein R₄ can be H, CH₃,Cl, Br, F, OCH₃, CF₃, an alkyl, a cycloalkyl, or an aryl; wherein R₅ canbe H, ═O, an alkyl, OR₇, NR₇, a heteroaryl, or an aryl, where R₇ can beH, an alkyl, or a heteroalkyl; and wherein the subject in need thereofcan be infected with or suspected of being infected with a pathogenicbacteria. The pathogenic bacteria can express beta lactamase. Thepathogenic bacteria can be resistant to at least one beta-lactamantibiotic. The method can further include the step of administering abeta lactam antibiotic to the subject in need thereof. The beta lactamantibiotic can be administered simultaneously or sequentially with thecomposition according to Formula 1.

In some aspect the composition can have a Formula according to Formula 2

wherein X can be O or N—R₉, wherein R₉ can be H, CH₃, an alkyl, or aheteroalkyl; wherein R₁ can be H, CH₃, Cl, Br, F, OCH₃, CF₃, an alkyl, acycloalkyl, or an aryl; wherein R₂ can be H, CH₃, Cl, Br, F, OCH₃, CF₃,alkyl, cycloalkyl, aryl, or R₂ and R₃, when taken together with theatoms to which they are attached, can form a cyclic structure having 5or 6 carbon atoms, one or more of which can be a heteroatom; wherein R₃can be H, CH₃, Cl, Br, F, OCH₃, CF₃, an alkyl, a cycloalkyl, or an aryl;wherein R₄ can be H, CH₃, Cl, Br, F, OCH₃, CF₃, an alkyl, a cycloalkyl,or an aryl; and wherein R₅ can be ═O, an alkyl, OR₇, NR₇, a heteroaryl,or an aryl, where R₇ can be H, an alkyl or a heteroalkyl.

In some aspects, the composition can have a Formula according to Formula3

wherein X can be CH, C, NH, N, N⁺,N⁺—O⁻, or NMe; wherein R₂ can be H,CH₃, Cl, Br, F, OCH₃, CF₃, an alkyl, a cycloalkyl, or an aryl; whereinR₃ can be H, CH₃, Cl, Br, F, OCH₃, CF₃, an alkyl, a cycloalkyl, or anaryl; and wherein R₄ can be H, CH₃, Cl, Br, F, OCH₃, CF₃, an alkyl, acycloalkyl, or an aryl.

In some aspects, the composition can have a Formula according to Formula4

wherein R₂ can be H, CH₃, Cl, Br, F, OCH₃, CF₃, an alkyl, a cycloalkyl,or an aryl; and wherein R₄ can be H, CH₃, Cl, Br, F, OCH₃, CF₃, analkyl, a cycloalkyl, or an aryl.

In some aspects, the composition can have a formula according to any oneof Formulas 5-19:

In some aspects, the composition can be included in a pharmaceuticalformulation.

In some aspects the composition can be administered orallyintravenously, intramuscularly, intravaginally, intraperitoneally,rectally, perenterally, topically, intranasally, or subcutaneously.

Also provided herein are compositions having a structure according toFormula 1, wherein X₁ can be O, O⁻, CH, or N—R₈, wherein R₈ can be H, analkyl, or a heteroalkyl; wherein X₂ can be PO(OH)₂, SO(OH)₂, CONHOH,COOH, or N(OH)COR₆, where R₆ can be H or CH₃; wherein X₃ can be H or SH;wherein X₄ can be CH, C, NH, or N⁺; wherein X₅ can be CH, or NH; whereinR₁ can be H, CH₃, Cl, Br, F, OCH₃, CF₃, an alkyl, a cycloalkyl, or anaryl; wherein R₂ can be H, CH₃, Cl, Br, F, OCH₃, CF₃, an alkyl, acycloalkyl, aryl, or R₂ and R₃, when taken together with the atoms towhich they are attached, can form a cyclic structure having 5 or 6carbon atoms, one or more of which can be a heteroatom; wherein R₃ canbe H, CH₃, Cl, Br, F, OCH₃, CF₃, an alkyl, a cycloalkyl, or an aryl;wherein R₄ can be H, CH₃, Cl, Br, F, OCH₃, CF₃, an alkyl, a cycloalkyl,or an aryl; and wherein R₅ is H, ═O, an alkyl, OR₇, NR₇, a heteroaryl,or an aryl, where R₇ can be H, an alkyl, or a heteroalkyl. In someaspects, the composition can have a structure according to any one ofFormulas 5-19. In some aspects, the composition can have a structureaccording to Formula 2, wherein X can be O or N—R₉, wherein R₉ can be H,CH₃, an alkyl, or a heteroalkyl; wherein R₁ can be H, CH₃, Cl, Br, F,OCH₃, CF₃, an alkyl, a cycloalkyl, or an aryl; wherein R₂ can be H, CH₃,Cl, Br, F, OCH₃, CF₃, alkyl, cycloalkyl, aryl, or R₂ and R₃, when takentogether with the atoms to which they are attached, can form a cyclicstructure having 5 or 6 carbon atoms, one or more of which can be aheteroatom; wherein R₃ can be H, CH₃, Cl, Br, F, OCH₃, CF₃, an alkyl, acycloalkyl, or an aryl; wherein R₄ can be H, CH₃, Cl, Br, F, OCH₃, CF₃,an alkyl, a cycloalkyl, or an aryl; and wherein R₅ can be ═O, an alkyl,OR₇, NR₇, a heteroaryl, or an aryl, where R₇ can be H, an alkyl, or aheteroalkyl. In some aspects, the composition can have a structureaccording to Formula 3, wherein X can be CH, C, NH, N, N⁺,N⁺—O⁻, or NMe;wherein R₂ can be H, CH₃, Cl, Br, F, OCH₃, CF₃, an alkyl, a cycloalkyl,or an aryl; wherein R₃ can be H, CH₃, Cl, Br, F, OCH₃, CF₃, an alkyl, acycloalkyl, or an aryl; and wherein R₄ can be H, CH₃, Cl, Br, F, OCH₃,CF₃, an alkyl, a cycloalkyl, or an aryl.

Also provided herein are compositions that can have a structureaccording to Formula 20

wherein X can be O or N; and R can be H, NH, NMe, or CH₃.

Also provided herein are pharmaceutical formulations that can include anamount of a composition according to any one of Formulas 1-20 asprovided herein and a pharmaceutically acceptable carrier. Thepharmaceutical formulations can further include a beta lactamantibiotic.

The amount of a composition to any one of Formulas 1-20 as providedherein can be an effective amount. The effective amount can increase theefficacy of a beta lactam antibiotic and/or reduce the amount of or theactivity of a beta lactamase.

BRIEF DESCRIPTION OF THE DRAWINGS

Further aspects of the present disclosure will be readily appreciatedupon review of the detailed description of its various embodiments,described below, when taken in conjunction with the accompanyingdrawings.

FIGS. 1A-1F show KPC-2 crystal structures of (FIG. 1A) 72875 (Formula5), (FIG. 1B) 73475 (Formula 8), (FIG. 1C) 72588 (Formula 6), (FIG. 1D)73043 (Formula 7), (FIG. 1E) 72674 (Formula 9), (FIG. 1F) 994284(Formula 11), and (FIG. 1G) 994942 (Formula 13).

FIGS. 2A-2H show crystal structures of NDM-1 bound with (FIG. 2A) 72875(Formula 5), (FIG. 2B) 73043 (Formula 7), (FIG. 2C) 994284 (Formula 11),(FIG. 2D) 994942 (Formula 13), (FIG. 2E) 994943 (Formula 14), (FIG. 2F)995016 (Formula 18), (FIG. 2G) 995018 (Formula 16), and (FIG. 2H)1014507 (Formula 17).

FIGS. 3A-3B show crystal structures of VIM-2 bound with (FIG. 3A) 995015(Formula 15) and (FIG. 3B) 995016 (Formula 18).

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the disclosure. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the disclosure, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the disclosure.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure, the preferredmethods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present disclosure is not entitled to antedate suchpublication by virtue of prior disclosure. Further, the dates ofpublication provided could be different from the actual publicationdates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwiseindicated, techniques of organic chemistry, biochemistry, pharmacology,botany, molecular biology, microbiology, medicinal chemistry and thelike, which are within the skill of the art. Such techniques areexplained fully in the literature.

Definitions

As used herein, “about,” “approximately,” and the like, when used inconnection with a numerical variable, generally refers to the value ofthe variable and to all values of the variable that are within theexperimental error (e.g., within the 95% confidence interval for themean) or within ±10% of the indicated value, whichever is greater.

As used herein, “control” is an alternative subject or sample used in anexperiment for comparison purposes and included to minimize ordistinguish the effect of variables other than an independent variable.A “control” can be positive or negative.

As used herein, “pharmaceutical formulation” refers to the combinationof an active agent, compound, or ingredient with a pharmaceuticallyacceptable carrier or excipient, making the composition suitable fordiagnostic, therapeutic, or preventive use in vitro, in vivo, or exvivo.

As used herein, “pharmaceutically acceptable carrier or excipient”refers to a carrier or excipient that is useful in preparing apharmaceutical formulation that is generally safe, non-toxic, and isneither biologically or otherwise undesirable, and includes a carrier orexcipient that is acceptable for veterinary use as well as humanpharmaceutical use. A “pharmaceutically acceptable carrier or excipient”as used in the specification and claims includes both one and more thanone such carrier or excipient.

As used herein, “pharmaceutically acceptable salt” refers to any acid orbase addition salt whose counter-ions are non-toxic to the subject towhich they are administered in pharmaceutical doses of the salts.

As used interchangeably herein, “subject,” “individual,” or “patient,”refers to a vertebrate, preferably a mammal, more preferably a human.Mammals include, but are not limited to, murines, simians, humans, farmanimals, sport animals, and pets. The term “pet” includes a dog, cat,guinea pig, mouse, rat, rabbit, ferret, and the like. The term farmanimal includes a horse, sheep, goat, chicken, pig, cow, donkey, llama,alpaca, turkey, and the like.

As used herein, “active agent” or “active ingredient” refers to acomponent or components of a composition to which the whole or part ofthe effect of the composition is attributed.

As used herein, “dose,” “unit dose,” or “dosage” refers to physicallydiscrete units suitable for use in a subject, each unit containing apredetermined quantity of the nanoparticle composition or formulationcalculated to produce the desired response or responses in associationwith its administration.

As used herein “immunomodulator,” refers to an agent, such as atherapeutic agent, which is capable of modulating or regulating one ormore immune function or response.

As used herein “anti-infectives” can include, but are not limited to,antibiotics, antibacterials, antifungals, antivirals, andantiproatozoals.

As used herein, “derivative” refers to any compound having the same or asimilar core structure to the compound but having at least onestructural difference, including substituting, deleting, and/or addingone or more atoms or functional groups. The term “derivative” does notmean that the derivative is synthesized from the parent compound eitheras a starting material or intermediate, although this may be the case.The term “derivative” can include prodrugs, or metabolites of the parentcompound. Derivatives include compounds in which free amino groups inthe parent compound have been derivatized to form amine hydrochlorides,p-toluene sulfoamides, benzoxycarboamides, t-butyloxycarboamides,thiourethane-type derivatives, trifluoroacetylamides,chloroacetylamides, or formamides. Derivatives include compounds inwhich carboxyl groups in the parent compound have been derivatized toform methyl and ethyl esters, or other types of esters, amides,hydroxamic acids, or hydrazides. Derivatives include compounds in whichhydroxyl groups in the parent compound have been derivatized to formO-acyl, O-carbamoyl, or O-alkyl derivatives. Derivatives includecompounds in which a hydrogen bond donating group in the parent compoundis replaced with another hydrogen bond donating group such as OH, NH, orSH. Derivatives include replacing a hydrogen bond acceptor group in theparent compound with another hydrogen bond acceptor group such asesters, ethers, ketones, carbonates, tertiary amines, imine, thiones,sulfones, tertiary amides, and sulfides. “Derivatives” also includesextensions of the replacement of the cyclopentane ring, as an example,with saturated or unsaturated cyclohexane or other more complex, e.g.,nitrogen-containing rings, and extensions of these rings with variousgroups.

As used herein, “administering” refers to an administration that isoral, topical, intravenous, subcutaneous, transcutaneous, transdermal,intramuscular, intra-joint, parenteral, intra-arteriole, intradermal,intraventricular, intracranial, intraperitoneal, intralesional,intranasal, rectal, vaginal, by inhalation, or via an implantedreservoir. The term “parenteral” includes subcutaneous, intravenous,intramuscular, intra-articular, intra-synovial, intrasternal,intrathecal, intrahepatic, intralesional, and intracranial injections orinfusion techniques.

The term “substituted” as used herein, refers to all permissiblesubstituents of the compounds described herein. In the broadest sense,the permissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,but are not limited to, halogens, hydroxyl groups, or any other organicgroupings containing any number of carbon atoms, e.g. 1-14 carbon atoms,and optionally include one or more heteroatoms such as oxygen, sulfur,or nitrogen grouping in linear, branched, or cyclic structural formats.Representative substituents include alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substitutedphenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy, substitutedphenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio,phenylthio, substituted phenylthio, arylthio, substituted arylthio,cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl,carboxyl, substituted carboxyl, amino, substituted amino, amido,substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid,phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl,polyaryl, substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀cyclic, heterocyclic, substituted heterocyclic, amino acid, peptide, andpolypeptide groups.

As used herein, “suitable substituent” means a chemically andpharmaceutically acceptable group, i.e., a moiety that does notsignificantly interfere with the preparation of or negate the efficacyof the inventive compounds. Such suitable substituents may be routinelychosen by those skilled in the art. Suitable substituents include butare not limited to the following: a halo, C₁-C₆ alkyl, C₂-C₆ alkenyl,C₁-C₆ haloalkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkoxy, C₂-C₆ alkynyl, C₃-C₈cycloalkenyl, (C₃-C₈ cycloalkyl)C₁-C₆ alkyl, (C₃-C₈ cycloalkyl)C₂-C₆alkenyl, (C₃-C₈ cycloalkyl)C₁-C₆ alkoxy, C₃-C₇ heterocycloalkyl, (C₃-C₇heterocycloalkyl)C₁-C₆ alkyl, (C₃-C₇ heterocycloalkyl) C₂-C₆ alkenyl,(C₃-C₇ heterocycloalkyl)C₁-C₆ alkoxyl, hydroxy, carboxy, oxo, sulfanyl,C₁-C₆ alkylsulfanyl, aryl, heteroaryl, aryloxy, heteroaryloxy, aralkyl,heteroaralkyl, aralkoxy, heteroaralkoxy, nitro, cyano, amino, C₁-C₆alkylamino, di-(C₁-C₆ alkyl)amino, carbamoyl, (C₁-C₆ alkyl)carbonyl,(C₁-C₆ alkoxy)carbonyl, (C₁-C₆ alkyl)aminocarbonyl, di-(C₁-C₆alkyl)aminocarbonyl, arylcarbonyl, aryloxycarbonyl, (C₁-C₆alkyl)sulfonyl, and arylsulfonyl. The groups listed above as suitablesubstituents are as defined hereinafter except that a suitablesubstituent may not be further optionally substituted.

As used herein, “optionally substituted” indicates that a group may beunsubstituted or substituted with one or more substituents as definedherein.

The term “alkyl” refers to the radical of saturated aliphatic groups(i.e., an alkane with one hydrogen atom removed), includingstraight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl(alicyclic) groups, alkyl-substituted cycloalkyl groups, andcycloalkyl-substituted alkyl groups.

In some embodiments, a straight chain or branched chain alkyl can have30 or fewer carbon atoms in its backbone (e.g., C₁-C₃₀ for straightchains, and C₃-C₃₀ for branched chains). In other embodiments, astraight chain or branched chain alkyl can contain 20 or fewer, 15 orfewer, or 10 or fewer carbon atoms in its backbone. Likewise, in someembodiments cycloalkyls can have 3-10 carbon atoms in their ringstructure. In some of these embodiments, the cycloalkyl can have 5, 6,or 7 carbons in the ring structure.

The term “alkyl” (or “lower alkyl”) as used herein is intended toinclude both “unsubstituted alkyls” and “substituted alkyls,” the latterof which refers to alkyl moieties having one or more substituentsreplacing a hydrogen on one or more carbons of the hydrocarbon backbone.Such substituents include, but are not limited to, halogen, hydroxyl,carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl),thiocarbonyl (such as a thioester, a thioacetate, or a thioformate),alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido,amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate,sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, oran aromatic or heteroaromatic moiety.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons in its backbone structure. Likewise, “lower alkenyl” and“lower alkynyl” have similar chain lengths.

It will be understood by those skilled in the art that the moietiessubstituted on the hydrocarbon chain can themselves be substituted, ifappropriate. For instance, the substituents of a substituted alkyl mayinclude halogen, hydroxy, nitro, thiols, amino, azido, imino, amido,phosphoryl (including phosphonate and phosphinate), sulfonyl (includingsulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, aswell as ethers, alkylthios, carbonyls (including ketones, aldehydes,carboxylates, and esters), —CF₃, —CN and the like. Cycloalkyls can besubstituted in the same manner.

The term “heteroalkyl,” as used herein, refers to straight or branchedchain, or cyclic carbon-containing radicals, or combinations thereof,containing at least one heteroatom. Suitable heteroatoms include, butare not limited to, O, N, Si, P, Se, B, and S, wherein the phosphorousand sulfur atoms are optionally oxidized, and the nitrogen heteroatom isoptionally quaternized. Heteroalkyls can be substituted as defined abovefor alkyl groups.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In preferred embodiments, the“alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl, and—S-alkynyl. Representative alkylthio groups include methylthio,ethylthio, and the like. The term “alkylthio” also encompassescycloalkyl groups, alkene and cycloalkene groups, and alkyne groups.“Arylthio” refers to aryl or heteroaryl groups. Alkylthio groups can besubstituted as defined above for alkyl groups.

The terms “alkenyl” and “alkynyl”, refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

The terms “alkoxyl” or “alkoxy,” as used herein, refers to an alkylgroup, as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl is an ether or resembles an alkoxyl, such as can berepresented by one of —O-alkyl, —O-alkenyl, and —O— alkynyl. The terms“aroxy” and “aryloxy”, as used interchangeably herein, can berepresented by —O-aryl or O-heteroaryl, wherein aryl and heteroaryl areas defined below. The alkoxy and aroxy groups can be substituted asdescribed above for alkyl.

The terms “amine” and “amino” (and its protonated form) areart-recognized and refer to both unsubstituted and substituted amines,e.g., a moiety that can be represented by the general formula:

wherein R, R′, and R″ each independently represent a hydrogen, an alkyl,an alkenyl, —(CH2)_(m)—R_(C) or R and R′ taken together with the N atomto which they are attached complete a heterocycle having from 4 to 8atoms in the ring structure; R_(C) represents an aryl, a cycloalkyl, acycloalkenyl, a heterocycle or a polycycle; and m is zero or an integerin the range of 1 to 8. In some embodiments, only one of R or R′ can bea carbonyl, e.g., R, R′ and the nitrogen together do not form an imide.In other embodiments, the term “amine” does not encompass amides, e.g.,wherein one of R and R′ represents a carbonyl. In further embodiments, Rand R′ (and optionally R″) each independently represent a hydrogen, analkyl or cycloakly, an alkenyl or cycloalkenyl, or alkynyl. Thus, theterm “alkylamine” as used herein means an amine group, as defined above,having a substituted (as described above for alkyl) or unsubstitutedalkyl attached thereto, i.e., at least one of R and R′ is an alkylgroup.

The term “amido” is art-recognized as an amino-substituted carbonyl andincludes a moiety that can be represented by the general formula:

wherein R and R′ are as defined above.

As used herein, “Aryl” refers to C₅-C₁₀-membered aromatic, heterocyclic,fused aromatic, fused heterocyclic, biaromatic, or bihetereocyclic ringsystems. Broadly defined, “aryl”, as used herein, includes 5-, 6-, 7-,8-, 9-, and 10-membered single-ring aromatic groups that may includefrom zero to four heteroatoms, for example, benzene, pyrrole, furan,thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine,pyrazine, pyridazine, pyrimidine, and the like. Those aryl groups havingheteroatoms in the ring structure may also be referred to as “arylheterocycles” or “heteroaromatics.” The aromatic ring can be substitutedat one or more ring positions with one or more substituents including,but not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl,cycloalkyl, hydroxyl, alkoxyl, amino (or quaternized amino), nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl,silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester,heterocyclyl, aromatic or heteroaromatic moieties, —CF₃, —CN, andcombinations thereof.

The term “aryl” also includes polycyclic ring systems having two or morecyclic rings in which two or more carbons are common to two adjoiningrings (i.e., “fused rings”) wherein at least one of the rings isaromatic, e.g., the other cyclic ring or rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls and/or heterocycles. Examples ofheterocyclic rings include, but are not limited to, benzimidazolyl,benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl,benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl,benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aHcarbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl,decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl,imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl,3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl,isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl,methylenedioxyphenyl, morpholinyl, naphthyridinyl,octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl,oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl,piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl,pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl,pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole,pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl,pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl,quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl,tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl,1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl,thienooxazolyl, thienoimidazolyl, thiophenyl, and xanthenyl. One or moreof the rings can be substituted as defined above for “aryl.”

The term “aralkyl,” as used herein, refers to an alkyl group substitutedwith an aryl group (e.g., an aromatic or heteroaromatic group).

The term “aralkyloxy” can be represented by —O-aralkyl, wherein aralkylis as defined above.

The term “carbocycle,” as used herein, refers to an aromatic ornon-aromatic ring(s) in which each atom of the ring(s) is carbon.

“Heterocycle” or “heterocyclic,” as used herein, refers to a monocyclicor bicyclic structure containing 3-10 ring atoms, and in someembodiments, containing from 5-6 ring atoms, wherein the ring atoms arecarbon and one to four heteroatoms each selected from the followinggroup of non-peroxide oxygen, sulfur, and N(Y) wherein Y is absent or isH, O, (C₁-C₁₀) alkyl, phenyl or benzyl, and optionally containing 1-3double bonds and optionally substituted with one or more substituents.Examples of heterocyclic rings include, but are not limited to,benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl,benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl,benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl,carbazolyl, 4aH carbazolyl, carbolinyl, chromanyl, chromenyl,cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,dihydrofuro[2,3 b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl,indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl,isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl,isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl,naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl,oxazolyl, oxepanyl, oxetanyl, oxindolyl, pyrimidinyl, phenanthridinyl,phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl,phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl,4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl,pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole,pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl,pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl,quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl,tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydropyranyl,tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl,1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl,thienooxazolyl, thienoimidazolyl, thiophenyl, and xanthenyl.Heterocyclic groups can optionally be substituted with one or moresubstituents at one or more positions as defined above for alkyl andaryl, for example, halogen, alkyl, aralkyl, alkenyl, alkynyl,cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate,phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic orheteroaromatic moiety, —CF₃, —CN, or the like.

The term “carbonyl” is art-recognized and includes such moieties as canbe represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R and R′are as defined above. Where X is an oxygen and R or R′ is not hydrogen,the formula represents an “ester”. Where X is an oxygen and R is asdefined above, the moiety is referred to herein as a carboxyl group, andparticularly when R is a hydrogen, the formula represents a “carboxylicacid.” Where X is an oxygen and R′ is hydrogen, the formula represents a“formate.” In general, where the oxygen atom of the above formula isreplaced by sulfur, the formula represents a “thiocarbonyl” group. WhereX is a sulfur and R or R′ is not hydrogen, the formula represents a“thioester.” Where X is a sulfur and R is hydrogen, the formularepresents a “thiocarboxylic acid.” Where X is a sulfur and R′ ishydrogen, the formula represents a “thioformate.” On the other hand,where X is a bond, and R is not hydrogen, the above formula represents a“ketone” group. Where X is a bond, and R is hydrogen, the above formularepresents an “aldehyde” group.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Exemplary heteroatoms include, but are notlimited to, boron, nitrogen, oxygen, phosphorus, sulfur, silicon,arsenic, and selenium. Heteroatoms, such as nitrogen, can have hydrogensubstituents and/or any permissible substituents of organic compoundsdescribed herein which satisfy the valences of the heteroatoms. It isunderstood that “substitution” or “substituted” includes the implicitproviso that such substitution is in accordance with permitted valenceof the substituted atom and the substituent, and that the substitutionresults in a stable compound, i.e., a compound that does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc.

As used herein, the term “nitro” refers to —NO₂; the term “halogen”designates —F, —Cl, —Br, or —I; the term “sulfhydryl” refers to —SH; theterm “hydroxyl” refers to —OH; and the term “sulfonyl” refers to —SO₂—.

As used herein, “effective amount” refers to the amount of a compositiondescribed herein or pharmaceutical formulation described herein thatwill elicit a desired biological or medical response of a tissue,system, animal, plant, protozoan, bacteria, yeast or human that is beingsought by the researcher, veterinarian, medical doctor or otherclinician. The desired biological response can be death, growthinhibition, reproductive inhibition, and/or development inhibition. Theeffective amount will vary depending on the exact chemical structure ofthe composition or pharmaceutical formulation, the causative agentand/or severity of the infection, disease, disorder, syndrome, orsymptom thereof being treated or prevented, the route of administration,the time of administration, the rate of excretion, the drug combination,the judgment of the treating physician, the dosage form, and the age,weight, general health, sex and/or diet of the subject to be treated.“Effective amount” can refer to an amount of a composition orpharmaceutical formulation described herein that can increase theefficacy of a beta lactam antibiotic and/or reduce amount or activity ofa beta lactamase

DISCUSSION

One of the most extensively studied bacterial resistance mechanisms isresistance to beta-lactam antibiotics such as pencillins,cephalosporins, and carbapenems. Beta-lactam antibiotics are among themost commonly prescribed antibiotics in the clinical setting.Beta-lactam antibiotics target the cross-linking of the bacterial cellwall, which ultimately results in cell death. Despite the numeroussuccesses of the beta-lactam antibiotics, bacteria have developedresistance to them, most often through the production of enzymes knownas beta-lactamases. Beta-lactamases are divided into four groups (classA, B, C, and D) based on their mechanism of action and amino acidsimilarity. Class A, C, and D beta-lactamases are serine enzymes thatuse an active site serine residue to hydrolyze the beta-lactam ring,whereas class B beta-lactamases are metallo-enzymes that use one or twozinc ions to carry out the hydrolysis reaction.

Since the introduction of benzylpenicillin in the early 1940s and theemergence of resistance a few years later, numerous semisyntheticbeta-lactam antibiotics such as cephalosporins and carbapenems weredeveloped that were more stable to beta-lactamase hydrolysis; however,as previously observed, resistance to these beta-lactam antibiotics isnow extensive.

Combining a beta-lactamase inhibitor with a beta-lactam antibiotic hasbeen the most successful approach in extending the efficacy ofbeta-lactam antibiotics; however, the clinically relevant beta-lactamaseinhibitors, clavulanic acid, sulbactam, and tazobactam are mosteffective only against class A beta-lactamases. In addition, theseinhibitors possess a beta-lactam ring, which can induce beta-lactamaseexpression and increase their susceptibility to hydrolysis. To addressthe prevalence of ESBLs and carbapenemases, as well as inhibitorresistant beta-lactamases, improved broad-spectrum beta-lactamaseinhibitors are urgently needed.

With that said, described herein are heterocyclic compounds,formulations, and that can inhibit beta-lactamase, such as KPC-2 andNDM-1. Use of a heterocyclic compound that can function as abroad-spectrum beta-lactamase inhibitor has had limited investigation.Because KPC-2 and NDM-1 beta-lactamases are implicated in multi-drugresistant infections, an inhibitor targeting these enzymes would be oftherapeutic interest. Also described herein are methods of making andusing the heterocyclic compounds and formulations described herein.Other compositions, compounds, methods, features, and advantages of thepresent disclosure will be or become apparent to one having ordinaryskill in the art upon examination of the following drawings, detaileddescription, and examples. It is intended that all such additionalcompositions, compounds, methods, features, and advantages be includedwithin this description, and be within the scope of the presentdisclosure.

Heterocyclic Compounds and Formulations Thereof

Heterocyclic Compounds

Provided herein are compounds according to Formula 1, where X₁ can be O,O⁻, CH, or N—R₈, wherein R₈ can be H, an alkyl, or a heteroalkyl; X₂ canbe PO(OH)₂, SO(OH)₂, CONHOH, COOH, or N(OH)COR₆, where R₆ can be H orCH₃, X₃ can be H or SH; X₄ can be CH, C, NH, or N⁺; X₅ can be CH, or NH;R₁ can be H, CH₃, Cl, Br, F, OCH₃, CF₃, an alkyl, a cycloalkyl, or anaryl; R₂ can be H, CH₃, Cl, Br, F, OCH₃, CF₃, an alkyl, a cycloalkyl,aryl, or R₂ and R₃, when taken together with the atoms to which they areattached, can form a cyclic structure having 5 or 6 carbon atoms, one ormore of which can be a heteroatom; R₃ can be H, CH₃, Cl, Br, F, OCH₃,CF₃, an alkyl, a cycloalkyl, or an aryl; R₄ can be H, CH₃, Cl, Br, F,OCH₃, CF₃, an alkyl, a cycloalkyl, or an aryl; R₅ can be H, ═O, analkyl, OR₇, NR₇, a heteroaryl, or an aryl, where R₇ can be H, an alkyl(such as a C₁-C₁₂ alkyl), or a heteroalkyl.

In some embodiments, the composition can have a formula according to anyone of Formulas 5-19. In embodiments, Formula 1, its derivatives, or anyof its substituents can be further substituted with a suitablesubstituent.

Also provided herein are compounds according to Formula 2, where X canbe O or N—R₉, wherein R₉ can be H, CH₃, an alkyl, or a heteroalkyl; R₁can be H, CH₃, Cl, Br, F, OCH₃, CF₃, an alkyl, a cycloalkyl, or an aryl;R₂ can be H, CH₃, Cl, Br, F, OCH₃, CF₃, alkyl, cycloalkyl, aryl, or R₂and R₃, when taken together with the atoms to which they are attached,can form a cyclic structure having 5 or 6 carbon atoms, one or more ofwhich can be a heteroatom; R₃ can be H, CH₃, Cl, Br, F, OCH₃, CF₃, analkyl, a cycloalkyl, or an aryl; R₄ can be H, CH₃, Cl, Br, F, OCH₃, CF₃,an alkyl, a cycloalkyl, or an aryl; and R₅ can be ═O, an alkyl, OR₇,NR₇, a heteroaryl, or an aryl, where R₇ can be H, an alkyl (such as aC₁-C₁₂ alkyl), or a heteroalkyl.

In embodiments, Formula 2, its derivatives, or any of its substituentscan be further substituted with a suitable substituent. In someembodiments, the compounds that can have a structure according toFormula 2 can be any one of compounds according to any one of Formulas5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, and 17.

Also provided herein are compounds according to Formula 3, where X canbe CH, C, NH, N, N⁺, N⁺—O⁻, or NMe, R₂ can be H, CH₃, Cl, Br, F, OCH₃,CF₃, an alkyl, a cycloalkyl, or an aryl; R₃ can be H, CH₃, Cl, Br, F,OCH₃, CF₃, an alkyl, a cycloalkyl, or an aryl; and R₄ can be H, CH₃, Cl,Br, F, OCH₃, CF₃, an alkyl, a cycloalkyl, or an aryl.

In embodiments, Formula 3, its derivatives, or any of its substituentscan be further substituted with a suitable substituent. In someembodiments, the compounds that can have a structure according toFormula 3 can be any one of compounds according to any one of Formulas18 or 19.

Also provided herein are compounds according to Formula 4, where R₂ canbe H, CH₃, Cl, Br, F, OCH₃, CF₃, an alkyl, a cycloalkyl, or an aryl andR₄ can be H, CH₃, Cl, Br, F, OCH₃, CF₃, an alkyl, a cycloalkyl, or anaryl.

In some embodiments, R₂ and R₄ can both be H. In embodiments, Formula 4,its derivatives, or any of its substituents can be further substitutedwith a suitable substituent. In some embodiments, the compounds that canhave a structure according to Formula 3 can be a compound according toFormulas 5.

Also provided herein are compounds that can have a structure accordingto Formula 20, where X can be O or N and R can be H, NH, NMe, or CH₃.

Methods of Making the Heterocyclic Compounds

The compounds described herein are commercially available or can besynthesized using techniques known to those skilled in the art.

Preparation of[(7-methoxy-5-methyl-2-oxo-2H-chromen-4-yl)methyl]phosphonic acid(Formula 11)

Step 1: Diethyl[(7-hydroxy-5-methyl-2-oxo-2H-chromen-4-yl)methyl]phosphonate. Ethyl4-(diethoxyphosphoryl)-3-oxobutanoate (0.250 g, 0.9 mmol) was added toconcentrated sulfuric acid (0.250 ml) with stirring at 0° C. To themixture was added 5-methylresorcinol (0.118 g, 0.9 mmol) and the mixturewas stirred at room temperature for 18 h. Cold water was then added tothe reaction mixture and the precipitate was collected by filtration,and dried to afford 230 mg of the product as a pale pink solid.

1H NMR: (400 MHz, CDCl₃) 6.56 (s, 1H), 6.32 (s, 1H), 6.05 (s, 1H), 4.15(q, J=7.3 Hz, 4H), 3.79 (s, 1H), 3.73 (s, 1H), 2.12 (s, 3H), 1.33 (t,J=6.8 Hz, 6H).

Step 2. Diethyl[(7-methoxy-5-methyl-2-oxo-2h-chromen-4-yl)methyl]phosphonate. Theproduct of step 1, diethyl[(7-hydroxy-5-methyl-2-oxo-2H-chromen-4-yl)methyl]phosphonate (0.020 g,0.1 mmol, 1.0 equiv.) was taken into methanol (0.3 mL) and treated with2M solution of (trimethylsilyl)diazomethane in hexanes (0.306 ml, 0.6mmol, 10.0 equiv.) and stirred at room temperature for 18 h. After thistime the reaction was 30% complete as judged by LC/MS. An additionalportion (20 eq) of (trimethylsilyl)diazomethane solution was added andthe reaction mixture stirred at room temperature for another 72 h. Thereaction mixture was then diluted with ethyl acetate and washed withsat.NaHCC₃, water & brine. The organic phase was dried over magnesiumsulfate, filtered, and concentrated to afford the crude product, whichwas purified by flash column chromatography (100% ethyl acetate/hexanesfollowed by 0-5% methanol/dichloromethane followed by 5%methanol/dichloromethane) to obtain 16 mg of the product as pale yellowsolid.

1H NMR: (400 MHz, CDCl₃) 6.8 (s, 1H), 6.59 (s, 1H), 6.18 (d, J=4.6 Hz,1H), 4.07-4.10 (m, 4H), 3.95 (s, 3H), 3.70-3.73 (m, 2H), 2.38 (s, 3H),1.26 (t, J=7.1 Hz, 6H).

Step 3. [(7-methoxy-5-methyl-2-oxo-2H-chromen-4-yl)methyl]phosphonicacid. To the product of Step 2, diethyl[(7-methoxy-5-methyl-2-oxo-2H-chromen-4-yl)methyl]phosphonate (0.013 g,0.0 mmol) was added a solution of 4M HCl in dioxane (0.4 mL) and thereaction mixture heated at 100° C. for 2 h. Another portion (0.2 mL) of4M HCl in dioxane was added and the mixture stirred at 100° C. foranother hour. The solution was then cooled to room temperature,concentrated, and triturated with ethyl acetate. The mixture wasvortexed & the ethyl acetate layer decanted. This procedure was repeatedthree times and the remaining solid was dried to obtain 6.2 mg of theproduct as a beige powder.

1H NMR: (400 MHz, d₆-DMSO) 6.8 (d, J=1.5 Hz, 2H), 6.12 (d, J=4.6 Hz,1H), 3.87 (s, 3H), 3.55-3.61 (m, 2H), 2.38 (s, 3H).

Pharmaceutical Formulations

Also provided herein are pharmaceutical formulations that can an amountof a heterocyclic compound or derivative thereof described herein in apharmaceutically acceptable carrier appropriate for administration to anindividual in need thereof. The individual in need thereof can have orcan be suspected of having a bacterial infection. The individual in needthereof can be or suspected of being infected with a bacteria that isresistant to one or more beta lactam antibiotics. Formulations can beadministered via any suitable administration route. For example, theformulations (and/or compositions) can be administered to the subject inneed thereof orally, intravenously, intramuscularly, intravaginally,intraperitoneally, rectally, parenterally, topically, intranasally, orsubcutaneously. Other suitable routes are described herein. Theheterocyclic compound(s) contained in the pharmaceutical formulation canhave a formula according to any one of Formulas 1-20 or a derivativethereof as set forth in the description and Examples provided herein.

Parenteral Formulations

The heterocyclic compounds and derivatives thereof can be formulated forparenteral delivery, such as injection or infusion, in the form of asolution or suspension. The heterocyclic compound(s) and/orderivative(s) thereof contained in the pharmaceutical formulation canhave a formula according to any one of Formulas 1-20, a derivativethereof encompassed by the description and Examples provided herein, ora pharmaceutical salt thereof. The formulation can be administered viaany route, such as, the blood stream or directly to the organ or tissueto be treated.

Parenteral formulations can be prepared as aqueous compositions usingtechniques is known in the art. Typically, such compositions can beprepared as injectable formulations, for example, solutions orsuspensions; solid forms suitable for using to prepare solutions orsuspensions upon the addition of a reconstitution medium prior toinjection; emulsions, such as water-in-oil (w/o) emulsions, oil-in-water(o/w) emulsions, and microemulsions thereof, liposomes, or emulsomes.

The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, one or more polyols (e.g., glycerol, propyleneglycol, and liquid polyethylene glycol), oils, such as vegetable oils(e.g., peanut oil, corn oil, sesame oil, etc.), and combinationsthereof. The proper fluidity can be maintained, for example, by the useof a coating, such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and/or by the use ofsurfactants. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride.

Solutions and dispersions of the heterocyclic compound(s) orderivative(s) thereof as described herein can be prepared in water oranother solvent or dispersing medium suitably mixed with one or morepharmaceutically acceptable excipients including, but not limited to,surfactants, dispersants, emulsifiers, pH modifying agents, andcombination thereof.

Suitable surfactants can be anionic, cationic, amphoteric or nonionicsurface active agents. Suitable anionic surfactants include, but are notlimited to, those containing carboxylate, sulfonate and sulfate ions.Suitable anionic surfactants include sodium, potassium, ammonium of longchain alkyl sulfonates and alkyl aryl sulfonates such as sodiumdodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodiumdodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodiumbis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodiumlauryl sulfate. Suitable cationic surfactants include, but are notlimited to, quaternary ammonium compounds such as benzalkonium chloride,benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzylammonium chloride, polyoxyethylene and coconut amine. Suitable nonionicsurfactants include ethylene glycol monostearate, propylene glycolmyristate, glyceryl monostearate, glyceryl stearate,polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG-150laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates,polyoxyethylene octylphenylether, PEG-1000 cetyl ether, polyoxyethylenetridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401,stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallowamide. Examples of amphoteric surfactants include sodiumN-dodecyl-β-alanine, sodium N-lauryl-β-iminodipropionate,myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.

The formulation can contain a preservative to prevent the growth ofmicroorganisms. Suitable preservatives include, but are not limited to,parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. Theformulation can also contain an antioxidant to prevent degradation ofthe heterocyclic compound or derivate thereof.

The formulation can be buffered to a pH of 3-8 for parenteraladministration upon reconstitution. Suitable buffers include, but arenot limited to, phosphate buffers, acetate buffers, and citrate buffers.

Water-soluble polymers can be used in the formulations for parenteraladministration. Suitable water-soluble polymers include, but are notlimited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, andpolyethylene glycol. Sterile injectable solutions can be prepared byincorporating the autophagic inhibitor or active derivative thereof inthe required amount in the appropriate solvent or dispersion medium withone or more of the excipients listed above, as required, followed byfiltered sterilization. Dispersions can be prepared by incorporating thevarious sterilized autophagic inhibitor or derivative thereof into asterile vehicle which contains the basic dispersion medium and therequired other ingredients from those listed above. Sterile powders forthe preparation of sterile injectable solutions can be prepared byvacuum-drying and freeze-drying techniques, which yields a powder of theautophagic inhibitor or active derivative thereof plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The powders can be prepared in such a manner that the particles areporous in nature, which can increase dissolution of the particles.Methods for making porous particles are well known in the art.

Pharmaceutical formulations for parenteral administration can be in theform of a sterile aqueous solution or suspension of particles formedfrom one or more heterocyclic compounds and/or derivatives thereof.Acceptable solvents include, for example, water, Ringer's solution,phosphate buffered saline (PBS), and isotonic sodium chloride solution.The formulation can also be a sterile solution, suspension, or emulsionin a nontoxic, parenterally acceptable diluent or solvent such as1,3-butanediol.

In some instances, the formulation can be distributed or packaged in aliquid form. In other embodiments, formulations for parenteraladministration can be packed as a solid, obtained, for example bylyophilization of a suitable liquid formulation. The solid can bereconstituted with an appropriate carrier or diluent prior toadministration.

Solutions, suspensions, or emulsions for parenteral administration canbe buffered with an effective amount of buffer necessary to maintain apH suitable for ocular administration. Suitable buffers include, but arenot limited to, acetate, borate, carbonate, citrate, and phosphatebuffers.

Solutions, suspensions, or emulsions for parenteral administration canalso contain one or more tonicity agents to adjust the isotonic range ofthe formulation. Suitable tonicity agents include, but are not limitedto, glycerin, mannitol, sorbitol, sodium chloride, and otherelectrolytes.

Solutions, suspensions, or emulsions for parenteral administration canalso contain one or more preservatives to prevent bacterialcontamination of the ophthalmic preparations. Suitable preservativesinclude, but are not limited to, polyhexamethylenebiguanidine (PHMB),benzalkonium chloride (BAK), stabilized oxychloro complexes (otherwiseknown as Purite®), phenylmercuric acetate, chlorobutanol, sorbic acid,chlorhexidine, benzyl alcohol, parabens, thimerosal, and mixturesthereof.

Solutions, suspensions, or emulsions for parenteral administration canalso contain one or more excipients, such as dispersing agents, wettingagents, and suspending agents.

Topical Formulations

The heterocyclic compounds and/or derivative(s) thereof as describedherein can be formulated for topical administration. The heterocycliccompound can have a formula according to any of Formulas 1-20. Suitabledosage forms for topical administration include creams, ointments,salves, sprays, gels, lotions, emulsions, liquids, and transdermalpatches. The formulation can be formulated for transmucosal,transepithelial, transendothelial, or transdermal administration. Thetopical formulations can contain one or more chemical penetrationenhancers, membrane permeability agents, membrane transport agents,emollients, surfactants, stabilizers, and combination thereof.

In some embodiments, the heterocyclic compounds and/or derivativesthereof can be administered as a liquid formulation, such as a solutionor suspension, a semi-solid formulation, such as a lotion or ointment,or a solid formulation. In some embodiments, the heterocyclic compoundsand/or derivatives thereof can be formulated as liquids, includingsolutions and suspensions, such as eye drops or as a semi-solidformulation, such as ointment or lotion for topical application to theskin, to the mucosa, such as the eye, to the vagina, or to the rectum.

The formulation can contain one or more excipients, such as emollients,surfactants, emulsifiers, penetration enhancers, and the like.

Suitable emollients include, without limitation, almond oil, castor oil,ceratonia extract, cetostearoyl alcohol, cetyl alcohol, cetyl esterswax, cholesterol, cottonseed oil, cyclomethicone, ethylene glycolpalmitostearate, glycerin, glycerin monostearate, glyceryl monooleate,isopropyl myristate, isopropyl palmitate, lanolin, lecithin, lightmineral oil, medium-chain triglycerides, mineral oil and lanolinalcohols, petrolatum, petrolatum and lanolin alcohols, soybean oil,starch, stearyl alcohol, sunflower oil, xylitol and combinationsthereof. In some embodiments, the emollients can be ethylhexylstearateand ethylhexyl palmitate.

Suitable surfactants include, but are not limited to, emulsifying wax,glyceryl monooleate, polyoxyethylene alkyl ethers, polyoxyethylenecastor oil derivatives, polysorbate, sorbitan esters, benzyl alcohol,benzyl benzoate, cyclodextrins, glycerin monostearate, poloxamer,povidone and combinations thereof. In some embodiments, the surfactantcan be stearyl alcohol.

Suitable emulsifiers include, but are not limited to, acacia, metallicsoaps, certain animal and vegetable oils, and various polar compounds,anionic emulsifying wax, calcium stearate, carbomers, cetostearylalcohol, cetyl alcohol, cholesterol, diethanolamine, ethylene glycolpalmitostearate, glycerin monostearate, glyceryl monooleate,hydroxpropyl cellulose, hypromellose, lanolin, hydrous, lanolinalcohols, lecithin, medium-chain triglycerides, methylcellulose, mineraloil and lanolin alcohols, monobasic sodium phosphate, monoethanolamine,nonionic emulsifying wax, oleic acid, poloxamer, poloxamers,polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives,polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates,propylene glycol alginate, self-emulsifying glyceryl monostearate,sodium citrate dehydrate, sodium lauryl sulfate, sorbitan esters,stearic acid, sunflower oil, tragacanth, triethanolamine, xanthan gumand combinations thereof. In some embodiments, the emulsifier can beglycerol stearate.

Suitable classes of penetration enhancers include, but are not limitedto, fatty alcohols, fatty acid esters, fatty acids, fatty alcoholethers, amino acids, phospholipids, lecithins, cholate salts, enzymes,amines and amides, complexing agents (liposomes, cyclodextrins, modifiedcelluloses, and diimides), macrocyclics, such as macrocylic lactones,ketones, and anhydrides and cyclic ureas, surfactants, N-methylpyrrolidones and derivatives thereof, DMSO and related compounds, ioniccompounds, azone and related compounds, and solvents, such as alcohols,ketones, amides, polyols (e.g., glycols).

Suitable emulsions include, but are not limited to, oil-in-water andwater-in-oil emulsions. Either or both phases of the emulsions caninclude a surfactant, an emulsifying agent, and/or a liquid non-volatilenon-aqueous material. In some embodiments, the surfactant can be anon-ionic surfactant. In other embodiments, the emulsifying agent is anemulsifying wax. In further embodiments, the liquid non-volatilenon-aqueous material is a glycol. In some embodiments, the glycol ispropylene glycol. The oil phase can contain other suitable oilypharmaceutically acceptable excipients. Suitable oily pharmaceuticallyacceptable excipients include, but are not limited to, hydroxylatedcastor oil or sesame oil can be used in the oil phase as surfactants oremulsifiers.

Lotions containing a heterocyclic compound and/or derivative thereof asdescribed herein are also provided. In some embodiments, the lotion canbe in the form of an emulsion having a viscosity of between 100 and 1000centistokes. The fluidity of lotions can permit rapid and uniformapplication over a wide surface area. Lotions can be formulated to dryon the skin leaving a thin coat of their medicinal components on theskin's surface.

Creams containing a heterocyclic compound and/or derivative thereof asdescribed herein are also provided. The cream can contain emulsifyingagents and/or other stabilizing agents. In some embodiments, the creamis in the form of a cream having a viscosity of greater than 1000centistokes, typically in the range of 20,000-50,000 centistokes.Creams, as compared to ointments, can be easier to spread and easier toremove.

One difference between a cream and a lotion is the viscosity, which isdependent on the amount/use of various oils and the percentage of waterused to prepare the formulations. Creams can be thicker than lotions,can have various uses, and can have more varied oils/butters, dependingupon the desired effect upon the skin. In some embodiments of a creamformulation, the water-base percentage can be about 60% to about 75% andthe oil-base can be about 20% to about 30% of the total, with the otherpercentages being the emulsifier agent, preservatives and additives fora total of 100%.

Ointments containing a heterocyclic compound and/or derivative thereofas described herein and a suitable ointment base are also provided.Suitable ointment bases include hydrocarbon bases (e.g., petrolatum,white petrolatum, yellow ointment, and mineral oil); absorption bases(hydrophilic petrolatum, anhydrous lanolin, lanolin, and cold cream);water-removable bases (e.g., hydrophilic ointment), and water-solublebases (e.g., polyethylene glycol ointments). Pastes typically differfrom ointments in that they contain a larger percentage of solids.Pastes are typically more absorptive and less greasy that ointmentsprepared with the same components.

Also described herein are gels containing a heterocyclic compound and/orderivative thereof as described herein, a gelling agent, and a liquidvehicle. Suitable gelling agents include, but are not limited to,modified celluloses, such as hydroxypropyl cellulose and hydroxyethylcellulose; carbopol homopolymers and copolymers; and combinationsthereof. Suitable solvents in the liquid vehicle include, but are notlimited to, diglycol monoethyl ether; alkylene glycols, such aspropylene glycol; dimethyl isosorbide; alcohols, such as isopropylalcohol and ethanol. The solvents can be selected for their ability todissolve the drug. Other additives, which can improve the skin feeland/or emolliency of the formulation, can also be incorporated. Suchadditives include, but are not limited, isopropyl myristate, ethylacetate, C₁₂-C₁₅ alkyl benzoates, mineral oil, squalane, cyclomethicone,capric/caprylic triglycerides, and combinations thereof.

Also described herein are foams that can include a heterocyclic compoundand/or derivative thereof as described herein. Foams can be an emulsionin combination with a gaseous propellant. The gaseous propellant caninclude hydrofluoroalkanes (HFAs). Suitable propellants include HFAssuch as 1,1,1,2-tetrafluoroethane (HFA 134a) and1,1,1,2,3,3,3-heptafluoropropane (HFA 227), but mixtures and admixturesof these and other HFAs that are currently approved or can becomeapproved for medical use are suitable. The propellants can be devoid ofhydrocarbon propellant gases, which can produce flammable or explosivevapors during spraying. Furthermore, the foams can contain no volatilealcohols, which can produce flammable or explosive vapors during use.

Buffers can be used to control pH of a composition. The buffers canbuffer the composition from a pH of about 4 to a pH of about 7.5, from apH of about 4 to a pH of about 7, or from a pH of about 5 to a pH ofabout 7. In some embodiments, the buffer can be triethanolamine.

Preservatives can be included to prevent the growth of fungi andmicroorganisms. Suitable preservatives include, but are not limited to,benzoic acid, butylparaben, ethyl paraben, methyl paraben,propylparaben, sodium benzoate, sodium propionate, benzalkoniumchloride, benzethonium chloride, benzyl alcohol, cetylpyridiniumchloride, chlorobutanol, phenol, phenylethyl alcohol, and thimerosal.

In certain embodiments, the formulations can be provided via continuousdelivery of one or more formulations to a patient in need thereof. Fortopical applications, repeated application can be done or a patch can beused to provide continuous administration of the noscapine analogs overan extended period of time.

Enteral Formulations

The heterocyclic compounds and/or derivatives thereof as describedherein can be prepared in enteral formulations, such as for oraladministration. The heterocyclic compound and/or derivative thereof asdescribed herein can be according to any of Formulas 1-20 as set forthherein, active derivative thereof, or pharmaceutical salt thereof.Suitable oral dosage forms include tablets, capsules, solutions,suspensions, syrups, and lozenges. Tablets can be made using compressionor molding techniques well known in the art. Gelatin or non-gelatincapsules can prepared as hard or soft capsule shells, which canencapsulate liquid, solid, and semi-solid fill materials, usingtechniques well known in the art.

Formulations containing a heterocyclic compound and/or derivativethereof as described herein can be prepared using pharmaceuticallyacceptable carriers. As generally used herein “carrier” includes, but isnot limited to, diluents, preservatives, binders, lubricants,disintegrators, swelling agents, fillers, stabilizers, and combinationsthereof. Polymers used in the dosage form include, but are not limitedto, suitable hydrophobic or hydrophilic polymers and suitable pHdependent or independent polymers. Suitable hydrophobic and hydrophilicpolymers include, but are not limited to, hydroxypropyl methylcellulose,hydroxypropyl cellulose, hydroxyethyl cellulose, carboxymethylcellulose, polyethylene glycol, ethylcellulose, microcrystallinecellulose, polyvinyl pyrrolidone, polyvinyl alcohol, polyvinyl acetate,and ion exchange resins. “Carrier” also includes all components of thecoating composition which can include plasticizers, pigments, colorants,stabilizing agents, and glidants.

Formulations containing a heterocyclic compound and/or derivativethereof as described herein can be prepared using one or morepharmaceutically acceptable excipients, including diluents,preservatives, binders, lubricants, disintegrators, swelling agents,fillers, stabilizers, and combinations thereof.

Delayed release dosage formulations containing a heterocyclic compoundand/or derivative thereof as described herein can be prepared asdescribed in standard references such as “Pharmaceutical dosage formtablets”, eds. Liberman et. al. (New York, Marcel Dekker, Inc., 1989),“Remington—The science and practice of pharmacy”, 20th ed., LippincottWilliams & Wilkins, Baltimore, Md., 2000, and “Pharmaceutical dosageforms and drug delivery systems”, 6th Edition, Ansel et al., (Media,Pa.: Williams and Wilkins, 1995). These references provide informationon excipients, materials, equipment and process for preparing tabletsand capsules and delayed release dosage forms of tablets, capsules, andgranules. These references provide information on carriers, materials,equipment and process for preparing tablets and capsules and delayedrelease dosage forms of tablets, capsules, and granules.

The formulations containing a heterocyclic compound and/or derivativethereof as described herein can be coated with a suitable coatingmaterial, for example, to delay release once the particles have passedthrough the acidic environment of the stomach. Suitable coatingmaterials include, but are not limited to, cellulose polymers such ascellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropylmethylcellulose, hydroxypropyl methylcellulose phthalate andhydroxypropyl methylcellulose acetate succinate; polyvinyl acetatephthalate, acrylic acid polymers and copolymers, and methacrylic resinsthat are commercially available under the trade name EUDRAGIT® (RothPharma, Westerstadt, Germany), zein, shellac, and polysaccharides.

Coatings can be formed with a different ratio of water soluble polymer,water insoluble polymers and/or pH dependent polymers, with or withoutwater insoluble/water soluble non polymeric excipient, to produce thedesired release profile. The coating can be performed on a dosage form(matrix or simple) which includes, but is not limited to, tablets(compressed with or without coated beads), capsules (with or withoutcoated beads), beads, particle compositions, “ingredient as is”formulated as, but not limited to, suspension form or as a sprinkledosage form.

Additionally, the coating material can contain conventional carrierssuch as plasticizers, pigments, colorants, glidants, stabilizationagents, pore formers and surfactants. Optional pharmaceuticallyacceptable excipients include, but are not limited to, diluents,binders, lubricants, disintegrants, colorants, stabilizers, andsurfactants.

Diluents, also referred to as “fillers,” can be used to increase thebulk of a solid dosage form so that a practical size is provided forcompression of tablets or formation of beads and granules. Suitablediluents include, but are not limited to, dicalcium phosphate dihydrate,calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose,microcrystalline cellulose, kaolin, sodium chloride, dry starch,hydrolyzed starches, pregelatinized starch, silicone dioxide, titaniumoxide, magnesium aluminum silicate and powdered sugar. The usualdiluents include inert powdered substances such as starches, powderedcellulose, especially crystalline and microcrystalline cellulose, sugarssuch as fructose, mannitol and sucrose, grain flours and similar ediblepowders. Typical diluents include, for example, various types of starch,lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic saltssuch as sodium chloride and powdered sugar. Powdered cellulosederivatives are also useful.

Binders can impart cohesive qualities to a solid dosage formulation, andthus can ensure that a tablet or bead or granule remains intact afterthe formation of the dosage forms. Suitable binder materials include,but are not limited to, starch, pregelatinized starch, gelatin, sugars(including sucrose, glucose, dextrose, lactose and sorbitol),polyethylene glycol, waxes, natural and synthetic gums such as acacia,tragacanth, sodium alginate, cellulose, includinghydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose,and veegum, and synthetic polymers such as acrylic acid and methacrylicacid copolymers, methacrylic acid copolymers, methyl methacrylatecopolymers, aminoalkyl methacrylate copolymers, polyacrylicacid/polymethacrylic acid and polyvinylpyrrolidone. Typical tabletbinders include substances such as starch, gelatin and sugars such aslactose, fructose, and glucose. Natural and synthetic gums, includingacacia, alginates, methylcellulose, and polyvinylpyrrolidone can also beused. Polyethylene glycol, hydrophilic polymers, ethylcellulose andwaxes can also serve as binders.

Lubricants can be included to facilitate tablet manufacture. Suitablelubricants include, but are not limited to, magnesium stearate, calciumstearate, stearic acid, glycerol behenate, polyethylene glycol, talc,and mineral oil. A lubricant can be included in a tablet formulation toprevent the tablet and punches from sticking in the die. The lubricantcan be chosen from such slippery solids as talc, magnesium and calciumstearate, stearic acid and hydrogenated vegetable oils.

Disintegrants can be used to facilitate dosage form disintegration or“breakup” after administration, and generally include, but are notlimited to, starch, sodium starch glycolate, sodium carboxymethylstarch, sodium carboxymethylcellulose, hydroxypropyl cellulose,pregelatinized starch, clays, cellulose, alginine, gums or cross linkedpolymers, such as cross-linked PVP (Polyplasdone® XL from GAF ChemicalCorp).

Stabilizers can be used to inhibit or retard drug decompositionreactions which include, by way of example, oxidative reactions.Suitable stabilizers include, but are not limited to, antioxidants,butylated hydroxytoluene (BHT); ascorbic acid, its salts and esters;Vitamin E, tocopherol and its salts; sulfites such as sodiummetabisulphite; cysteine and its derivatives; citric acid; propylgallate, and butylated hydroxyanisole (BHA).

Additional Active Agents

In some embodiments, an amount of one or more additional active agentsare included in the pharmaceutical formulation containing a heterocycliccompound and/or derivative thereof as described herein, a derivativethereof, or pharmaceutical salt thereof. Suitable additional activeagents include, but are not limited to, antipyretics, immunomodulators,analgesics, and anti-infectives. In some embodiments, the additionalactive agent is a B-lactam antibiotic. Suitable beta-lactam antibioticsare described below.

Suitable antipyretics include, but are not limited to, non-steroidalanti-inflammants (e.g. ibuprofen, naproxen, ketoprofen, and nimesulide),aspirin and related salicylates (e.g. choline salicylate, magnesiumsalicylae, and sodium salicaylate), paracetamol/acetaminophen,metamizole, nabumetone, phenazone, and quinine.

Suitable immunomodulators include, but are not limited to, prednisone,azathioprine, 6-MP, cyclosporine, tacrolimus, methotrexate, interleukins(e.g. IL-2, IL-7, and IL-12), cytokines (e.g. interferons (e.g. IFN-α,IFN-β, IFN-ε, IFN-κ, IFN-ω, and IFN-γ), granulocyte colony-stimulatingfactor, and imiquimod), chemokines (e.g. CCL3, CCL26 and CXCL7),cytosine phosphate-guanosine, oligodeoxynucleotides, glucans,antibodies, and aptamers).

Suitable analgesics include, but are not limited to,paracetamol/acetaminophen, non-steroidal anti-inflammants (e.g.ibuprofen, naproxen, ketoprofen, and nimesulide), COX-2 inhibitors (e.g.rofecoxib, celecoxib, and etoricoxib), opioids (e.g. morphine, codeine,oxycodone, hydrocodone, dihydromorphine, pethidine, buprenorphine),tramadol, norepinephrine, flupiretine, nefopam, orphenadrine,pregabalin, gabapentin, cyclobenzaprine, scopolamine, methadone,ketobemidone, piritramide, and aspirin and related salicylates (e.g.choline salicylate, magnesium salicylae, and sodium salicaylate).

Suitable anti-infectives include, but are not limited to, amebicides(e.g. nitazoxanide, paromomycin, metronidazole, tnidazole, chloroquine,and iodoquinol), aminoglycosides (e.g. paromomycin, tobramycin,gentamicin, amikacin, kanamycin, and neomycin), anthelmintics (e.g.pyrantel, mebendazole, ivermectin, praziquantel, abendazole,miltefosine, thiabendazole, oxamniquine), antifungals (e.g. azoleantifungals (e.g. itraconazole, fluconazole, posaconazole, ketoconazole,clotrimazole, miconazole, and voriconazole), echinocandins (e.g.caspofungin, anidulafungin, and micafungin), griseofulvin, terbinafine,flucytosine, and polyenes (e.g. nystatin, and amphotericin b),antimalarial agents (e.g. pyrimethamine/sulfadoxine,artemether/lumefantrine, atovaquone/proquanil, quinine,hydroxychloroquine, mefloquine, chloroquine, doxycycline, pyrimethamine,and halofantrine), antituberculosis agents (e.g. aminosalicylates (e.g.aminosalicylic acid), isoniazid/rifampin,isoniazid/pyrazinamide/rifampin, bedaquiline, isoniazid, ethanmbutol,rifampin, rifabutin, rifapentine, capreomycin, and cycloserine),antivirals (e.g. amantadine, rimantadine, abacavir/lamivudine,emtricitabine/tenofovir,cobicistat/elvitegravir/emtricitabine/tenofovir,efavirenz/emtricitabine/tenofovir, avacavir/lamivudine/zidovudine,lamivudine/zidovudine, emtricitabine/tenofovir,emtricitabine/opinavir/ritonavir/tenofovir, interferonalfa-2v/ribavirin, peginterferon alfa-2b, maraviroc, raltegravir,dolutegravir, enfuvirtide, foscarnet, fomivirsen, oseltamivir,zanamivir, nevirapine, efavirenz, etravirine, rilpiviirine,delaviridine, nevirapine, entecavir, lamivudine, adefovir, sofosbuvir,didanosine, tenofovir, avacivr, zidovudine, stavudine, emtricitabine,xalcitabine, telbivudine, simeprevir, boceprevir, telaprevir,lopinavir/ritonavir, fosamprenvir, dranuavir, ritonavir, tipranavir,atazanavir, nelfinavir, amprenavir, indinavir, sawuinavir, ribavirin,valcyclovir, acyclovir, famciclovir, ganciclovir, and valganciclovir),carbapenems (e.g. doripenem, meropenem, ertapenem, andcilastatin/imipenem), cephalosporins (e.g. cefadroxil, cephradine,cefazolin, cephalexin, cefepime, ceflaroline, loracarbef, cefotetan,cefuroxime, cefprozil, loracarbef, cefoxitin, cefaclor, ceftibuten,ceftriaxone, cefotaxime, cefpodoxime, cefdinir, cefixime, cefditoren,cefizoxime, and ceftazidime), glycopeptide antibiotics (e.g. vancomycin,dalbavancin, oritavancin, and telvancin), glycylcyclines (e.g.tigecycline), leprostatics (e.g. clofazimine and thalidomide),lincomycin and derivatives thereof (e.g. clindamycin and lincomycin),macrolides and derivatives thereof (e.g. telithromycin, fidaxomicin,erthromycin, azithromycin, clarithromycin, dirithromycin, andtroleandomycin), linezolid, sulfamethoxazole/trimethoprim, rifaximin,chloramphenicol, fosfomycin, metronidazole, aztreonam, bacitracin, betalactam antibiotics (benzathine penicillin (benzatihine andbenzylpenicillin), phenoxymethylpenicillin, cloxacillin, flucoxacillin,methicillin, temocillin, mecillinam, azlocillin, mezlocillin,piperacillin, amoxicillin, ampicillin, bacampicillin, carbenicillin,piperacillin, ticarcillin, amoxicillin/clavulanate,ampicillin/sulbactam, piperacillin/tazobactam, clavulanate/ticarcillin,penicillin, procaine penicillin, oxacillin, dicloxacillin, nafcillin,cefazolin, cephalexin, cephalosporin C, cephalothin, cefaclor,cefamandole, cefuroxime, cefotetan, cefoxitin, cefiximine, cefotaxime,cefpodoxime, ceftazidime, ceftriaxone, cefepime, cefpirome, ceftaroline,biapenem, doripenem, ertapenem, faropenem, imipenem, meropenem,panipenem, razupenem, tebipenem, thienamycin, azrewonam, tigemonam,nocardicin A, taboxinine, and beta-lactam), quinolones (e.g.lomefloxacin, norfloxacin, ofloxacin, qatifloxacin, moxifloxacin,ciprofloxacin, levofloxacin, gemifloxacin, moxifloxacin, cinoxacin,nalidixic acid, enoxacin, grepafloxacin, gatifloxacin, trovafloxacin,and sparfloxacin), sulfonamides (e.g. sulfamethoxazole/trimethoprim,sulfasalazine, and sulfasoxazole), tetracyclines (e.g. doxycycline,demeclocycline, minocycline, doxycycline/salicyclic acid,doxycycline/omega-3 polyunsaturated fatty acids, and tetracycline), andurinary anti-infectives (e.g. nitrofurantoin, methenamine, fosfomycin,cinoxacin, nalidixic acid, trimethoprim, and methylene blue).

Methods of Using the Heterocyclic Compounds and Formulations Thereof

The heterocyclic compounds and formulations thereof described herein canbe administered to a subject in need thereof. The subject in needthereof can be infected with or be suspected of being infected withpathogenic bacteria. The subject in need thereof can be symptomatic orasymptomatic.

In embodiments, the amount of the heterocyclic compounds or formulationsthereof delivered to the subject in need thereof can be an amountsufficient to inhibit beta lactamase and/or increase the effectivenessof a co-administered beta-lactam antibiotic (i.e. an effective amount).It will be appreciated that co-administered can refer to a beta-lactamantibiotic that is included in the formulation or provided in a dosageform separate from the heterocyclic compound or formulation thereof. Theeffective amount of the heterocyclic compound or formulation thereof,such as those described herein, can range from about 1 mg/kg to about500 mg/kg. In some embodiments, the effective amount ranges from about10 mg/kg to about 100 mg/kg. If further embodiments, the effectiveamount ranges from about 1 mg to about 1000 mg. In some embodiments, theeffective amount can be about 500 mg to about 1000 mg.

Administration of the heterocyclic compounds, derivatives thereof,and/or formulations thereof can be systemic or localized. The compoundsand formulations described herein can be administered to the subject inneed thereof one or more times per day. In an embodiment, thecompound(s) and/or formulation(s) thereof can be administered oncedaily. In some embodiments, the compound(s) and/or formulation(s)thereof can be administered given once daily. In another embodiment, thecompound(s) and/or formulation(s) thereof can be administered isadministered twice daily. In some embodiments, when administered, aneffective amount of the compounds and/or formulations are administeredto the subject in need thereof. The compound(s) and/or formulation(s)thereof can be administered one or more times per week. In someembodiments the compound(s) and/or formulation(s) thereof can beadministered 1 day per week. In other embodiments, the compound(s)and/or formulation(s) thereof can be administered 2 to 7 days per week.

In some embodiments, the compound(s) and/or formulation(s) thereof, canbe administered in a dosage form. The amount or effective amount of thecompound(s) and/or formulation(s) thereof can be divided into multipledosage forms. For example, the effective amount can be split into twodosage forms and the one dosage forms can be administered, for example,in the morning, and the second dosage form can be administered in theevening. Although the effective amount is given over two doses, in oneday, the subject receives the effective amount. In some embodiments theeffective amount is about 1 to about 1000 mg per day. The effectiveamount in a dosage form can range from about 1 mg/kg to about 1000mg/kg. The dosage form can be formulated for oral, vaginal, intravenous,transdermal, subcutaneous, intraperitoneal, or intramuscularadministration. Preparation of dosage forms for various administrationroutes are described elsewhere herein.

EXAMPLES

Now having described the embodiments of the present disclosure, ingeneral, the following Examples describe some additional embodiments ofthe present disclosure. While embodiments of the present disclosure aredescribed in connection with the following examples and thecorresponding text and figures, there is no intent to limit embodimentsof the present disclosure to this description. On the contrary, theintent is to cover all alternatives, modifications, and equivalentsincluded within the spirit and scope of embodiments of the presentdisclosure.

Example 1

Expression and Purification of Beta-Lactamases. For his tag KPC-2beta-lactamase, bacteria were grown overnight at 30 C with shaking in 50mL LB broth supplemented with 50 μg/mL kanamycin. Two liters of LB brothsupplemented with 50 μg/mL kanamycin, 200 mM sorbitol, and 5 mM betainewere each inoculated with 10 mL of overnight bacterial culture. Cultureswere then grown at 37 C until an optical density at 600 nm (OD₆₀₀) of0.6-0.7. Protein expression was then initiated by the addition of IPTG(final concentration 0.5 mM), followed by growth for 16 hr at 20 C.Cells were pelleted by centrifugation and stored at −80 C until furtheruse. The his tag KPC-2 beta-lactamase was purified by nickel affinitychromatography and gel filtration. Briefly, the cell pellets were thawedand re-suspended in 40 mL of buffer A (20 mM Tris-HCl pH 8.0, 300 mMNaCl, 20 mM imidazole) with one complete protease inhibitor cocktailtablet (Roche) and disrupted by sonication, followed byultracentrifugation to clarify the lysate. After ultracentrifugation,the supernatant was passed through a 0.22 μm filter before loading ontoa 5 mL HisTrap HP affinity column (GE Healthcare Life Sciences, USA)pre-equilibrated with buffer A. His tag KPC-2 was eluted by a linearimidazole gradient (20 mM to 500 mM). Fractions were analyzed bySDS-PAGE. Fractions containing his tag KPC-2 were concentrated using a10 k NMWL Amicon Ultra-15 Centrifugal Filter Unit. Concentrated his tagKPC-2 was then loaded onto a superdex 75 gel filtration column (GEHealthcare Life Sciences) pre-equilibrated with 20 mM Tris-HCl pH 8.0,300 mM NaCl. Protein concentration was determined by absorbance at 280using an extinction coefficient of 39,545. SDS-PAGE analysis indicatedthat the eluted protein was more than 95% pure.

For sumo tag NDM-1 metallo-beta-lactamase, bacteria were grown overnightat 30 C with shaking in 50 mL LB broth supplemented with 100 μg/mLampicillin. Two liters of LB broth supplemented with 100 μg/mLampicillin were each inoculated with 10 mL of overnight bacterialculture. Cultures were then grown at 37 C until an optical density at600 nm (OD600) of 0.6-0.7. Protein expression was then initiated by theaddition of IPTG (final concentration 0.5 mM), followed by growth for 16hr at 20 C. Cells were pelleted by centrifugation and stored at −80 Cuntil further use. The sumo tag NDM-1 beta-lactamase was purified bynickel affinity chromatography and gel filtration. Briefly, the cellpellets were thawed and re-suspended in 40 mL of buffer A (20 mM HEPESpH 7.4, 0.5 M NaCl, 20 mM imidazole) with one complete proteaseinhibitor cocktail tablet (Roche) and disrupted by sonication, followedby ultracentrifugation to clarify the lysate. After ultracentrifugation,the supernatant was passed through a 0.22 μm filter before loading ontoa 5 mL HisTrap HP affinity column (GE Healthcare Life Sciences, USA)pre-equilibrated with buffer A. Sumo tag NDM-1 was eluted by a linearimidazole gradient (20 mM to 500 mM). Fractions were analyzed bySDS-PAGE. Fractions containing sumo tag NDM-1 were buffer exchanged into20 mM HEPES pH 7.0, 100 mM NaCl. Cleavage of the sumo tag was thencarried out with ULP1 protease overnight at room temperature and thenconcentrated using a 10 k NMWL Amicon Ultra-15 Centrifugal Filter Unit.The sample was then loaded back onto a nickel affinity column and theflow through was collected, containing the untag NDM-1. NDM-1 wasconcentrated and loaded onto a gel filtration column (GE Healthcare LifeSciences) pre-equilibrated with 20 mM HEPES pH 7.0, 100 mM NaCl. Proteinconcentration was determined by absorbance at 280 using an extinctioncoefficient of 27,960. SDS-PAGE analysis indicated that the elutedprotein was more than 95% pure.

Steady-State Kinetic Analysis. Steady-state kinetic parameters weredetermined by using a Biotek Cytation Multi-Mode Reader. For KPC-2, eachassay was performed in 100 mM Tris-HCl pH 7.0, 0.01% Triton X-100 at 37C. V_(max) and K_(m) were determined from initial steady-statevelocities from nitrocefin read at a wavelength of 486 nm. The kineticparameters were obtained using the non-linear portion of the data to theHenri-Michaelis (equation 1) using SigmaPlot 12.5.V=Vmax[S]/(K _(m)+[S])  Equation (1)IC₅₀, defined as the inhibitor concentration that results in a 50%reduction of nitrocefin (20 μm) hydrolysis was determined bymeasurements of initial velocities after mixing 1 nM of KPC-2 withincreasing concentrations of inhibitors. The inhibition constant (K) wascalculated according to equation 2:K _(i)=IC₅₀/([S]/K _(n)+1)  Equation (2)For NDM-1, the procedures were the same as above except the assay wasperformed in 100 mM Tris-HCl pH 7.0, 1 μM ZnSO₄, 0.01% Triton X-100.Steady-state kinetic parameters are shown in Table 1. Table 1demonstrates inhibition of KPC-2 and NDM-1 by compounds of the disclosedherein. Compounds according to Formulas 5-10 were purchased fromcommercial sources. The last compound was de novo synthesized.

TABLE 1 K_(i) K_(i) (KPC- (NDM- His-Tag His-tag Compound 2) 1) KPC-2 KiNDM-1 Ki VIM-2 Ki SHV-2 Ki Number Structure (μM) (μM) (μM) (μM) (μM)(μM) 72875 Formula 13.4  5 72588 Formula 1.5 ~100  6 73043 Formula 26.6 7 73475 Formula 13.5  8 72674 Formula 154.4  9 73152 Formula 39.5 10994284 Formula 5.7 11 994942 Formula 0.4881 54.97 1.8 No 13 inhibition994943 Formula 122 9.12 14 995015 Formula 8.9 4.7 15 995016 Formula447.1 24.33 18 995018 Formula 79.2 15.57 16 995019 Formula precipitationprecipitation precipitation precipitation 19 1014507 Fornula 4.22 17MIC Studies. Compounds were tested for synergy with the carbapenemantibiotic, imipenem, against BL21(DE3) E. coli expressing KPC-2. MICvalues were determined with the Mueller-Hinton broth microdilutionmethod. To test for inhibitory activity, compounds were dissolved inDMSO and dilutions were carried out using LB broth. Compounds were testat 100 □M with increasing concentrations of imipenem. A control wasperformed with DMSO to demonstrate that DMSO did not have an effect onbacteria growth. After inoculation, the plates are incubated at 37 C for24 hours. The MIC of the compounds were then determined visually. MICdata are shown in Tables 2 and 3. Tables 2 and 3 demonstrate MICs ofimipenem when combined with select coumarin phosphonate derivatives. ForTable 3, 16 μg/mL of compounds were tested for synergy with thecarbapenem antibiotic, imipenem, against a K. pneumoniae strainexpressing KPC-2.

TABLE 2 MIC (μg/mL) Imi- Imi- Imi- Imi- Imi- penem + penem + penem +penem + Strain penem 72588 72875 73043 73475 E. coli 4 0.5 1 1 1BL21(DE3) pET-GST- bla_(KPC-2)

TABLE 3 MIC (μg/mL) 73043 72588 IMP/ IMP/ (For- (For- For- For- Phe-mula mula mula mula Organism ATCC notype 7) 6) IMP 7 6 K. 4683KPC-2 >32 >32 8 8 4 pneumoniaeCrystallization and Soaking Experiments. Crystallization trials werecarried out by using Qiagen crystallization kits. An initial conditionwas found in the Classics suite A4 containing 2 M Ammonium sulfate and5% (v/v) isopropanol. Crystals of his tag KPC-2 were grown at 20 C usingthe hanging-drop vapor diffusion in EasyXtal 15-Well tools (Qiagen).Protein solutions (10-20 mg/ml) in 20 mM Tris-HCl pH 8.0, 300 mM NaClwere mixed 1:2 (v/v) with a reservoir solution containing 2 M ammoniumsulfate and either 5% (v/v) isopropanol or 5% (v/v) ethanol. Droplets(1.5 □L) were microseeded with 0.5 μL of diluted seed stock. Crystalstypically began to form within two weeks. To obtain the inhibitor boundstructures, KPC-2 crystals were soaked in a solution containing 1.44 Msodium citrate and 10 mM of coumarin phosphonate derivative for 1 hour.The soaked crystals were cryo-protected in a solution containing 1.15 Msodium citrate, 20% (v/v) glycerol, and 0.5 mM coumarin phosphonatederivative and flash-frozen in liquid nitrogen.

Data Collection and Structure Determinations. Data for the KPC-2 complexstructures were collected at the Advanced Photon Source (APS) beamline22-ID-D and at the Advanced Light Source (ALS) beamline 8.3.1.Diffraction data were indexed and integrated with iMosflm and scaledwith SCALA from the CCP4 suite. Phasing was performed using molecularreplacement with the program Phaser with the truncated KPC-2 structure(PDB 3C5A). Structure refinement was performed using phenix.refine andmodel building in WinCoot. The program eLBOW in Phenix was used toobtain geometry restraint information for the coumarin phosphonatederivatives. The final model qualities were assessed using MolProbity.The structures of select compounds are shown in FIGS. 1A-3B.

We claim:
 1. A compound according to Formula 11


2. A pharmaceutical formulation comprising: an amount of a compoundaccording to Formula 11

 and a pharmaceutically acceptable carrier.
 3. The pharmaceuticalformulation of claim 2, further comprising a beta lactam antibiotic. 4.The pharmaceutical formulation of claim 2, wherein the compoundaccording to Formula 11 is present in the formulation at an amounteffective to increase the efficacy of a beta lactam antibiotic or reducethe amount of or the activity of a beta lactamase.
 5. A method oftreating a pathogenic bacterial infection in a subject in need thereof,the method comprising: administering, to the subject in need thereofhaying a pathogenic bacterial infection, an amount of a compoundaccording to Formula 11


6. The method of claim 5, wherein the pathogenic bacterial infection iscaused by a pathogenic bacteria is resistant to at least one beta-lactamantibiotic.
 7. The method of claim 5, further comprising administering abeta lactam antibiotic to the subject in need thereof.